Application of trichosanthes kirilowii tkwrky37 gene in improving plant anthracnose resistance

Abstract

This invention discloses the application of the *Trichosanthes kirilowii* TkWRKY37 gene in enhancing anthracnose resistance in plants, belonging to the field of genetic engineering. This invention is the first to isolate and clone the TkWRKY37 gene, which is specifically highly expressed in anthracnose-resistant *Trichosanthes kirilowii* varieties. Its nucleotide sequence is shown in SEQ ID NO.1, and it encodes a protein with the amino acid sequence shown in SEQ ID NO.2. Using *Arabidopsis thaliana* as a background, this invention constructed *Arabidopsis thaliana* plants overexpressing the TkWRKY37 gene. Anthracnose infection and phenotypic observation experiments were conducted on wild-type and transgenic plants. The results showed that the *Arabidopsis thaliana* plants overexpressing the TkWRKY37 gene exhibited significantly enhanced disease resistance and a significantly higher survival rate; indicating that the TkWRKY37 gene positively regulates anthracnose resistance in plants and can be used for genetic improvement, providing technical support for *Trichosanthes kirilowii* resistance breeding and production applications.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly to the application of Trichosanthes kirilowii TkWRKY37 gene in improving the resistance of plants to anthracnose. Background Art

[0002] Trichosanthes kirilowii Trichosanthes kirilowii Maxim.) is a perennial vine of the genus Trichosanthes in the Cucurbitaceae family and is a commonly used traditional Chinese medicine. In recent years, with the discovery of the health care and edible value of Trichosanthes kirilowii seeds, the market demand for Trichosanthes kirilowii has increased rapidly, the planting area has been continuously expanded, and there is a steady development trend. Since Trichosanthes kirilowii mainly reproduces asexually in daily production, with the increase of planting years, the degeneration of seed traits is serious, and the occurrence of diseases increases year by year, resulting in the reduction of the yield and quality of Trichosanthes kirilowii, seriously affecting production. At present, there are common problems in Trichosanthes kirilowii production, such as frequent occurrence of pests and diseases and few high-resistance varieties. During the growth process, anthracnose can infect the whole plant, causing irreversible damage to Trichosanthes kirilowii and even leading to plant death, ultimately resulting in a reduction in production. Therefore, it is necessary to explore genes related to yield.

[0003] Transgenic technology refers to artificially transferring one or several known functional genes of an organism into another organism to settle down, so that the organism obtains new functions. Through transgenic technology, beneficial genes can be transferred into crops to make them have stronger abilities such as disease resistance, insect resistance, drought resistance, etc., thereby increasing the yield of crops. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of Trichosanthes kirilowii TkWRKY37 gene in improving the resistance of plants to anthracnose, so as to solve the problems existing in the above-mentioned prior art. The present invention firstly discovers that Trichosanthes kirilowii TkWRKY37 gene positively regulates the resistance of plants to anthracnose, which can be used for genetic improvement, providing technical support for the resistance breeding and production application of Trichosanthes kirilowii.

[0005] To achieve the above purpose, the present invention provides the following scheme: The present invention provides an application of Trichosanthes kirilowii TkWRKY37 protein in any one of the following: (1) Application in regulating the resistance of plants to anthracnose; (2) Application in cultivating transgenic plants with improved resistance to anthracnose; (3) Application in preparing products for improving the resistance of plants to anthracnose; The amino acid sequence of the Trichosanthes kirilowii TkWRKY37 protein is as shown in SEQ ID NO.2.

[0006] Furthermore, by upregulating the expression level of the coding gene of the Trichosanthes kirilowii TkWRKY37 protein in plants, the resistance of the plants to anthracnose can be improved; The plant is Arabidopsis thaliana or Trichosanthes kirilowii.

[0007] This invention also provides an application of the Trichosanthes kirilowii TkWRKY37 gene in any of the following: (1) Application in regulating plant resistance to anthracnose; (2) Application in the cultivation of transgenic plants with enhanced resistance to anthracnose; (3) Application in the preparation of products that enhance plant resistance to anthracnose; The nucleotide sequence of the Trichosanthes kirilowii TkWRKY37 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the expression level of the Trichosanthes kirilowii TkWRKY37 gene was upregulated in the plant to enhance the plant's resistance to anthracnose. The plant in question is Arabidopsis thaliana or Trichosanthes kirilowii.

[0009] The present invention also provides an application of a recombinant vector, wherein the recombinant vector comprises the Trichosanthes kirilowii TkWRKY37 gene; The nucleotide sequence of the Trichosanthes kirilowii TkWRKY37 gene is shown in SEQ ID NO.1; The application is any one of the following: (1) Application in regulating plant resistance to anthracnose; (2) Application in the cultivation of transgenic plants with enhanced resistance to anthracnose; (3) Application in the preparation of products that enhance plant resistance to anthracnose.

[0010] The present invention also provides the use of engineered bacteria comprising the above-described recombinant vector in any of the following: (1) Application in regulating plant resistance to anthracnose; (2) Application in the cultivation of transgenic plants with enhanced resistance to anthracnose; (3) Application in the preparation of products that enhance plant resistance to anthracnose.

[0011] The present invention also provides a method for improving plant resistance to anthracnose, comprising the step of upregulating the expression level of the Trichosanthes kirilowii TkWRKY37 gene in the plant to improve the plant's resistance to anthracnose; The nucleotide sequence of the Trichosanthes kirilowii TkWRKY37 gene is shown in SEQ ID NO.1.

[0012] Furthermore, the method for upregulating the expression level of the Trichosanthes kirilowii TkWRKY37 gene includes overexpressing the Trichosanthes kirilowii TkWRKY37 gene in plants; The plant in question is Arabidopsis thaliana or Trichosanthes kirilowii.

[0013] This invention also provides a method for breeding plants with enhanced resistance to anthracnose, comprising the following steps: The Trichosanthes kirilowii TkWRKY37 gene was overexpressed in plant cells, the plant cells were then cultured, and the plant cells were used to regenerate plants, thus obtaining the plant with enhanced resistance to anthracnose. The nucleotide sequence of the Trichosanthes kirilowii TkWRKY37 gene is shown in SEQ ID NO.1.

[0014] Furthermore, the plant in question is Arabidopsis thaliana or Trichosanthes kirilowii.

[0015] The present invention discloses the following technical effects: This invention marks the first isolation and cloning of the TkWRKY37 gene, which is specifically highly expressed in anthracnose-resistant Trichosanthes kirilowii varieties. Its nucleotide sequence is shown in SEQ ID NO.1, and it encodes a protein with the amino acid sequence shown in SEQ ID NO.2. Using Arabidopsis thaliana as a background, this invention constructed Arabidopsis thaliana plants overexpressing the TkWRKY37 gene. Anthracnose infection and phenotypic observation experiments were conducted on wild-type and transgenic plants. The results showed that the Arabidopsis thaliana plants overexpressing the TkWRKY37 gene exhibited significantly enhanced disease resistance and a significantly higher survival rate. This indicates that the TkWRKY37 gene positively regulates anthracnose resistance in plants and can be used for genetic improvement, providing technical support for the breeding and production application of Trichosanthes kirilowii. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The graph shows the difference in TkWRKY37 gene expression after inoculation with pathogens in different resistant Trichosanthes kirilowii varieties in Example 1. Figure 2 The plasmid map of PMDC43 in Example 1; Figure 3 Phenotypic characteristics of TkWRKY37 transgenic Arabidopsis plants before and after inoculation with anthracnose. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] This invention involves inoculating different anthracnose-resistant Trichosanthes kirilowii varieties with anthracnose pathogen, detecting gene expression levels, and screening for a differentially expressed Trichosanthes kirilowii gene, TkWRKY37. The statistical results of its expression level are shown below. Figure 1 As shown.

[0024] The CDS sequence of the Trichosanthes kirilowii gene TkWRKY37 is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0025] SEQ ID NO.1: 。

[0026] SEQ ID NO.2: MDIFLDLNLDPASSYANSAKDEALDSSKREFEAGEIYWDKEKLSLSLANKGSDLNPTLEEELDRKIKENGKLSQMLRAMYEKYMNLHKQVMYLLSQQKQNSEIEAVSRKRKADGEEEYENLEGICSTRDEDFNRWLKRPRLNGNSKVSKVFVQKDASDP SLVVKDGYQWRKYGQKVTRDNPSPRAYFKCSSAPNCPVKKKVQRSLEDPTILVATYEGEHSHASHFQTELSLRSINGGKGSAVLATIKPSCATVTLDLIHEDGLFKSPKDYASSESAVWQELLVQQMASSLKKDPEFAGIVAGAISGQVLGNQTNRE.

[0027] Example 1: Construction of Arabidopsis thaliana overexpression vector The Arabidopsis overexpression vector used was pMDC43, and the vector recombination was performed using enzyme digestion and ligation. The Arabidopsis was infected using the flower dip method. In the Arabidopsis overexpression experiment, to facilitate subsequent screening, the infected wild-type Col-0 Arabidopsis was usually referred to as the T0 generation, the seeds obtained after infection were called T1 generation seeds, the seedlings that germinated from the T1 generation seeds were called T1 generation seedlings, and the seeds harvested from the T1 generation seedlings were called T2 generation seeds. Genotypic segregation occurred in the T2 generation seeds, and homozygous positive seedlings were selected at the T2 generation. Different gene expression levels were measured to obtain overexpression lines with different gene expression levels.

[0028] 1. Trichosanthes kirilowii TkWRKY37 gene amplification Using the octoploid Trichosanthes kirilowii 'Wanlou 17' cDNA as a template, primers were designed using Vector NT1 based on the published gene CDS sequences in the database. Primers were also designed using homologous recombination based on the vector's multiple cloning site, and vector sequence adapters were added. The resulting primers have nucleotide sequences as shown in SEQ ID NO. 3-4.

[0029] TkWRKY37 -DC43-FP: ATGGATGAACTATACAAAGGGATGGATATTTTTCTTGGACCTTAATTTG, SEQ ID NO.3; TkWRKY37 -DC43-RP: AACATATCCAGTCACTATGGGTTATTCTCTGTTTGTTTGGTTTCCC, SEQ ID NO.4; PCR was performed using the high-fidelity enzyme KOD-plus-Neo in a 25 μL volume. The amplification reaction mixture consisted of: 2 mM dNTPs 2 μL, 25 mM MgSO4 1.5 μL, Buffer 2 μL, upstream primer 1 μL, downstream primer 1 μL, ddH2O 14.5 μL, cDNA template 1.5 μL, and enzyme 0.5 μL.

[0030] The PCR amplification reaction procedure uses a two-step method common to KOD enzymes because the primers have adapters and a high Tm value. The specific steps are as follows: 98℃ for 5 min; 98℃ for 30 s, 68℃ for 1 kb / min, 35 cycles; 72℃ for 10 min; 20℃ for 1 h.

[0031] After the reaction is complete, add an appropriate amount of DNA loading buffer, and use 1% agarose gel electrophoresis to determine the size of the product and recover the DNA fragments with the correct bands. The recovery steps are as follows: (1) Cut the DNA agarose fragment containing the target gene under UV light, put it into a 2.0 mL centrifuge tube, calculate the gel weight, estimate the gel volume according to 100 mg = 100 μL, and then perform gel recovery according to the Axygen gel recovery kit. (2) Add 4 gel volumes of DE-A solution to a centrifuge tube, mix well, and heat in a 75°C water bath until the gel is completely melted. This process takes about 8-10 minutes. (3) Add 1 / 2 volume of Buffer DE-A to Buffer DE-B, mix thoroughly until the solution turns bright yellow; (4) Transfer the mixture from step (3) to a 2.0 mL centrifuge tube with a preparation tube, centrifuge at 12000 rpm for 1 min, and discard the filtrate; (5) Place the preparation tube back into the centrifuge tube, add 500 μL of W1 solution, centrifuge at 12000 rpm for 30 s, and discard the filtrate; (6) Place the preparation tube back into the centrifuge tube, add 700 μL of W2 solution, centrifuge at 12000 rpm for 30 s, and discard the filtrate.

[0032] (7) Place the preparation tube back into the centrifuge tube and centrifuge at 12,000 rpm for 2 min. Let stand at room temperature for 2-5 min to allow the ethanol to evaporate completely; (8) Place the preparation tube back into a clean 1.5 mL centrifuge tube, add 26 μL of preheated deionized water at 65°C to the center of the membrane, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, discard the preparation tube, and take 3-5 μL of the recovered product for agarose gel electrophoresis to detect whether the recovery was successful.

[0033] 2. Carrier Construction 2.1 Linearized Cloning Vector Based on the primer design, the pMDC43 empty vector was selected for double digestion with AscⅠ and SalⅠ (as shown in the image). Figure 2 (As shown), 37℃, 5 h, the enzyme digestion system is as follows: pMDC43 plasmid 14 μL, ddH2O 2.4 μL, CutSmart Buffer 2 μL, Asc Ⅰ 0.8 μL, Sal I. 0.8 μL, total volume 20 μL.

[0034] 2.2 Homologous recombination reaction The linearized vector after enzyme digestion was detected by 1% agarose gel electrophoresis. The vector backbone was recovered and recombined with the gene PCR product. The system consisted of 2 μL of 5×CE MultiS Buffer, 3 μL of linearized cloning vector, 4 μL of PCR product, and Exnase. ® MultiS 1 μL, total volume 10 μL.

[0035] After the system is prepared, gently pipette the components to mix them. Incubate at 37°C for 2 hours. After completion, place on ice for 5 minutes.

[0036] 2.3 Transformation of recombinant products, plating, and culture shaking. Transfer 5 μL of the recombinant product to 50 μL of competent E. coli (DH5α) cells. Gently agitate the bottom of the tube, place it on ice for 30 min, heat shock at 42°C for 90 s, then place it on ice for another 2 min. Add 100 μL of antibiotic-free LB medium, incubate at 37°C and 180 rpm for 1 h. Spread 100 μL of the bacterial culture evenly onto a kanamycin-resistant plate, invert the plate, and incubate overnight at 37°C for 10–12 h. Once single colonies have grown on the plate, use a sterile inoculation loop to inoculate the single colony from the antibiotic-resistant plate into 2 mL of liquid LB medium containing an appropriate amount of kanamycin resistance. Incubate at 37°C and shake at 220 rpm for 10–12 h. After turbidity, extract the plasmid.

[0037] 3. Plasmid extraction Collect the turbid bacterial culture in a 2.0 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, and then extract the plasmid according to the Axygen plasmid extraction kit.

[0038] (1) Add 250 μL of Buffer S1 solution to suspend the bacterial precipitate. The suspension must be uniform and no small bacterial clumps should be left. (2) Add 250 μL Buffer S2, gently invert 5-7 times to mix the liquid thoroughly and promote the complete lysis of the cells until a clear solution is formed; (3) Add 350 μL Buffer S3, mix thoroughly and gently by inverting the container 7-10 times, and centrifuge at 12000 rpm for 10 min at room temperature; (4) Take about 750 μL of the supernatant from step (3) and transfer it to a 2.0 mL centrifuge tube containing the preparation tube. Centrifuge at 12000 rpm at room temperature for 1 min and discard the filtrate. (5) Place the preparation tube back into the centrifuge tube, add 500 μL Buffer W1, let stand for 1 min, centrifuge at 12000 rpm for 1 min, and discard the filtrate; (6) Place the preparation tube back into the centrifuge tube, add 750 μL of Buffer W2, let stand for 1 min, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Add another 750 μL of Buffer W2 in the same way, wash once, and discard the filtrate; (7) Place the preparation tube back into the 2.0 mL centrifuge tube and centrifuge at 12000 rpm for 1 min; (8) Transfer the preparation tube to a new 1.5 mL centrifuge tube, and add 70 μL of deionized water (preheated to 65°C beforehand to improve elution efficiency) to the center of the membrane in the preparation tube. Let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min. Discard the preparation tube, collect the filtrate, check the plasmid concentration, and store it at -20°C for later use.

[0039] 4. Preparation of Agrobacterium competent cells (GV3010) (1) Prepare 1.0 L LB liquid culture medium and 1.6 L 10% glycerol, sterilize and set aside; (2) Streak single GV3101 competent cells on LB solid medium containing three antibiotics (rifampin, gentamicin, and tetracycline) and incubate in the dark at 28°C for about 48 h. (3) After the colonies grow, pick a single colony and inoculate it into 5 mL of LB liquid medium containing three types of resistance. Incubate overnight at 28°C and 220 rpm for about 48 h. (4) Inoculate 5 mL of the overnight culture into 500 mL of antibiotic-free LB liquid medium and incubate at 28°C and 220 rpm for about 6-8 h until OD is reached. 600 It is 0.8; (5) Collect the bacterial culture in a 50 mL sterilized centrifuge tube, centrifuge at 4000 rpm and 4℃ for 5 min, and collect the bacterial cells; (6) Wash and resuspend the bacterial cells with 40 mL of 10% sterile glycerol, centrifuge at 4000 rpm and 4℃ for 5 min, and discard the supernatant; (7) Repeat step (6) once; (8) In the last step of glycerol washing, when the supernatant is discarded, resuspend GV3101 with the refluxed glycerol, dispense 50 μL into 1.5 mL centrifuge tubes (which need to be sterilized in advance), and quickly place them in liquid nitrogen at -80℃ for later use.

[0040] 5. Recombinant plasmid transformed Agrobacterium GV3101 (1) Take GV3101 Agrobacterium competent cells out of the -80℃ freezer, place them on ice to thaw, add 1 μL of recombinant plasmid, and add the mixture to a pre-prepared electroporation cup; (2) 2500 V, 1-2 electric shocks; (3) Add 400 μL of non-resistant LB solution to the electroporation cup, mix well, and then transfer the mixture thoroughly to a 1.5 mL centrifuge tube; (4) Shake at 28℃, 220 rpm, in the dark for 3-5 h; (5) Take 50 μL and spread it on LB plates containing kanamycin, rifampin, gentamicin and tetracycline resistance, and incubate in the dark at 28℃ for 48h; (6) Once the bacterial spots have grown, shake the bacteria to propagate them and store them for later use.

[0041] 6. Genetic transformation in Arabidopsis thaliana 6.1 Sterilization treatment of Arabidopsis thaliana seeds (1) Take an appropriate amount of Arabidopsis thaliana seeds into a 2.0 mL centrifuge tube, add 12% Bleach in a clean bench and treat for 10 min, during which the centrifuge tube is constantly inverted and shaken to ensure that the seeds are in full contact with the disinfectant. (2) Remove the 12% Bleach solution from the centrifuge tube, and wash it with sterile water 6-8 times, shaking thoroughly for 1-2 minutes each time; (3) After washing, add an appropriate amount of sterile water and place the Arabidopsis seeds at 4°C in the dark for 3 days to vernalize.

[0042] 6.2 Cultivation of wild-type Arabidopsis thaliana (1) After vernalization, the seeds were evenly sown on 1 / 2 MS solid medium and placed vertically in an artificial climate chamber for growth. They were cultured at 25°C under 16 h light / 8 h darkness conditions. (2) Mix vermiculite and sieved black soil after high pressure sterilization in a volume ratio of 3:1, and divide them into small square basins (7 cm × 7 cm × 10 cm). Place the square basins in a tray and add tap water to the bottom of the tray to allow the nutrient soil and vermiculite to slowly soak and moisten. (3) When the Arabidopsis thaliana grows to 6-8 days and the root length is about 6 cm, open the culture dish and gently transplant the Arabidopsis thaliana into the prepared nutrient soil with tweezers. Be careful not to damage the root system. Press down the roots of the Arabidopsis thaliana with an appropriate amount of water and cover with plastic wrap to prevent the seedlings from losing moisture. (4) After a week, when the seedlings have grown steadily, the plastic wrap can be removed to allow them to grow normally. Water and fertilize them at appropriate times to prevent and control pests and diseases. (5) When Arabidopsis thaliana flowers, it is soaked in the solution.

[0043] 6.3 Agrobacterium infection in Arabidopsis thaliana (1) After activating the Agrobacterium tumefaciens containing the recombinant plasmid, take 500 μL to 50 mL of liquid LB medium containing four antibiotics (kanamycin, rifampin, gentamicin, tetracycline) for expansion culture, and culture in the dark at 28℃ and 220 rpm for 36-48 h. (2) Collect the bacterial culture in a 50 mL centrifuge tube, centrifuge at 6000 rpm for 10 min at room temperature, and discard the supernatant; (3) Add 15 mL of Arabidopsis thaliana transformation buffer, fully suspend the bacterial cells and mix well; (4) Use a Pasteur dropper to draw a certain amount of suspension and drip it onto the stigma of Arabidopsis thaliana that is about to flower. After the infection is finished, cover the infected plant with a black plastic bag and remove it after 24 hours. (5) One week later, repeat the above dyeing steps to improve conversion efficiency; (6) After the two inoculations are completed, water and fertilize in a timely manner according to the growth status of Arabidopsis thaliana, and prevent and control diseases and pests. (7) When most of the siliques of the infected Arabidopsis thaliana mature and turn yellow, stop watering and harvest the T0 generation transgenic seeds one after another.

[0044] 6.4 Screening of transgenic positive lines of Arabidopsis thaliana (1) T0 generation transgenic Arabidopsis seeds were disinfected, vernalized, and then evenly sown on 1 / 2 MS solid medium containing 25 mg / L hygromycin. They were placed vertically in an artificial climate chamber and cultured at 25°C under 16 h light / 8 h darkness conditions. (2) After growing in the greenhouse for 7-10 days, Arabidopsis thaliana that can grow normally on the plate is a transgenic positive seedling. Because the fusion plasmid contains a GFP tag, take 1 cm from the root tip and observe it under a fluorescence microscope. If the cell nucleus contains a green fluorescent signal, it is preliminarily identified as a positive seedling and transplanted into nutrient soil. (3) When Arabidopsis thaliana is about to flower, take 1-2 leaves from each plant and extract DNA for PCR verification. Refer to the instructions of the Sangon Biotech kit for DNA extraction steps. (4) PCR amplification of the target gene fragment. PCR was performed using a common mixed enzyme. The reaction volume was 25 μL. The amplification reaction volume was as follows: enzyme 12.5 μL, upstream primer 1 μL, downstream primer 1 μL, ddH2O 8.5 μL, cDNA template 2 μL. The PCR amplification reaction program was as follows: 96℃ for 5 min; 96℃ for 15 s, 56℃ for 15 s, 68℃ for 1 min / kb, 36 cycles; 72℃ for 10 min; 20℃ for 2 h.

[0045] After the reaction was complete, the size of the product was determined by 1% agarose gel electrophoresis.

[0046] (5) Screening of homozygous lines with different expression levels Leaves from six homozygous Arabidopsis thaliana lines were selected, and nine samples were taken from each line. RNA was extracted from the leaves and reverse transcribed into cDNA. The gene expression levels of different lines were measured by RT-PCR. Homozygous lines with different expression levels were screened out. Finally, the three lines with the highest expression levels were selected as OE-7, OE-11, and OE-13.

[0047] Example 2: Phenotypic observation before and after anthrax inoculation Transgenic and wild-type Arabidopsis seeds were germinated under the same conditions and planted in an artificial climate chamber with suitable temperature, light, and humidity. Anthracnose was inoculated into the seedlings when they reached the 5-leaf stage, and phenotypes were observed. The survival rate of the Arabidopsis plants was then recorded.

[0048] The results are as follows Figure 3 As shown, anthracnose was tested on transgenic plants and wild-type controls. Colletotrichum (spp.) Inoculation experiment. Transgenic lines exhibited a significantly enhanced disease resistance phenotype, specifically a significant reduction in lesion area and disease severity. This result indicates that the TkWRKY37 gene positively regulates Arabidopsis resistance to anthracnose and may play a key role in the signal transduction or effector mechanisms of plant defense responses.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a Trichosanthes kirilowii TkWRKY37 protein in any of the following: (1) Application in regulating plant resistance to anthracnose; (2) Application in the cultivation of transgenic plants with enhanced resistance to anthracnose; (3) Application in the preparation of products that enhance plant resistance to anthracnose; The amino acid sequence of the Trichosanthes kirilowii TkWRKY37 protein is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, Upregulating the expression level of the gene encoding the TkWRKY37 protein of Trichosanthes kirilowii in plants enhances the plant's resistance to anthracnose. The plant in question is Arabidopsis thaliana or Trichosanthes kirilowii.

3. An application of the Trichosanthes kirilowii TkWRKY37 gene in any of the following: (1) Application in regulating plant resistance to anthracnose; (2) Application in the cultivation of transgenic plants with enhanced resistance to anthracnose; (3) Application in the preparation of products that enhance plant resistance to anthracnose; The nucleotide sequence of the Trichosanthes kirilowii TkWRKY37 gene is shown in SEQ ID NO.

1.

4. The application as described in claim 3, characterized in that, Upregulating the expression level of the TkWRKY37 gene in *Trichosanthes kirilowii* in plants enhances the plant's resistance to anthracnose. The plant in question is Arabidopsis thaliana or Trichosanthes kirilowii.

5. An application of a recombinant vector, characterized in that, The recombinant vector includes the Trichosanthes kirilowii TkWRKY37 gene; The nucleotide sequence of the Trichosanthes kirilowii TkWRKY37 gene is shown in SEQ ID NO.1; The application is any one of the following: (1) Application in regulating plant resistance to anthracnose; (2) Application in the cultivation of transgenic plants with enhanced resistance to anthracnose; (3) Application in the preparation of products that enhance plant resistance to anthracnose.

6. The use of an engineered bacterium comprising the recombinant vector of claim 5 in any of the following: (1) Application in regulating plant resistance to anthracnose; (2) Application in the cultivation of transgenic plants with enhanced resistance to anthracnose; (3) Application in the preparation of products that enhance plant resistance to anthracnose.

7. A method for improving plant resistance to anthracnose, characterized in that, This includes the step of upregulating the expression level of the Trichosanthes kirilowii TkWRKY37 gene in plants to improve the resistance of said plants to anthracnose; The nucleotide sequence of the Trichosanthes kirilowii TkWRKY37 gene is shown in SEQ ID NO.

1.

8. The method as described in claim 7, characterized in that, The method for upregulating the expression level of the Trichosanthes kirilowii TkWRKY37 gene includes overexpressing the Trichosanthes kirilowii TkWRKY37 gene in plants; The plant in question is Arabidopsis thaliana or Trichosanthes kirilowii.

9. A breeding method for plants with enhanced resistance to anthracnose, characterized in that, Includes the following steps: The Trichosanthes kirilowii TkWRKY37 gene was overexpressed in plant cells, the plant cells were then cultured, and the plant cells were used to regenerate plants, thus obtaining the plant with enhanced resistance to anthracnose. The nucleotide sequence of the Trichosanthes kirilowii TkWRKY37 gene is shown in SEQ ID NO.

1.

10. The breeding method as described in claim 9, characterized in that, The plant in question is Arabidopsis thaliana or Trichosanthes kirilowii.

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